The impact of mRNA poly(A) tail length on eukaryotic translation stages

Abstract

The poly(A) tail plays an important role in maintaining mRNA stability and influences translation efficiency via binding with PABP. However, the impact of poly(A) tail length on mRNA translation remains incompletely understood. This study explores the effects of poly(A) tail length on human translation. We determined the translation rates in cell lysates using mRNAs with different poly(A) tails. Cap-dependent translation was stimulated by the poly(A) tail, however, it was largely independent of poly(A) tail length, with an exception observed in the case of the 75 nt poly(A) tail. Conversely, cap-independent translation displayed a positive correlation with poly(A) tail length. Examination of translation stages uncovered the dependence of initiation and termination on the presence of the poly(A) tail, but the efficiency of initiation remained unaffected by poly(A) tail extension. Further study unveiled that increased binding of eRFs to the ribosome with the poly(A) tail extension induced more efficient hydrolysis of peptidyl-tRNA. Building upon these findings, we propose a crucial role for the 75 nt poly(A) tail in orchestrating the formation of a double closed-loop mRNA structure within human cells which couples the initiation and termination phases of translation.

Graphical Abstract

Figure 1.

Effect of poly(A) tail length on translation in HEK293F cell lysate. (A) Schematic representation of the model mRNAs encoding firefly luciferase (Fluc) used in cell-free translation. (B) Initial translation rate v₀ of capped and uncapped Fluc mRNA in the absence and the presence of exogenous PABP (PABP_exo). RLU, relative luminescent units. The graphs show the mean ± standard error, n = 3. Asterisks indicate statistically significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001), insignificant differences are not shown. Red asterisks indicate differences between the values of different curves, blue and orange asterisks indicate differences with the A0 value of the corresponding curve. (C) Western blot analysis of the HEK 293F lysate with antibodies specific to human eRF1, eRF3 and PABP.

Figure 2.

Effect of poly(A) tail length on 48S preinitiation complex formation. (  A) Schematic representation of the model mRNAs encoding MVHL tetrapeptide used in the reconstitution of ribosomal complexes. (B) Relative amount of the 48S preinitiation complexes formed in the reconstituted mammalian translation system in the presence or absence of PABP on model capped and uncapped mRNAs with different poly(A) tails (0–100). The graphs show the mean ± standard error, n = 4. The amount of the 48S formed on capped mRNA without a poly(A) tail was set as 1. Asterisks indicate statistically significant differences between the values of different curves (*, P < 0.05; **, P < 0.01; ***, P < 0.001), while insignificant differences are not shown.

Figure 3.

Effect of poly(A) tail length on elongation and post-termination complexes formation. (A) Toe-printing analysis of the 80S initiation complex and preTC formation in the reconstituted mammalian translation system in the presence or absence of PABP on the model mRNA with 75 nt poly(A) tail (MVHL A75). The graph shows the mean ± standard error, n = 4. Asterisks indicate statistically significant differences (**, P < 0.01), while insignificant differences are not shown. (B) Toe-printing analysis of the postTC formation in the reconstituted mammalian translation system in the presence of PABP and different amounts of release factors on the model mRNAs without poly(A) tail (MVHL A0) and with 75 nt poly(A) tail (MVHL A75). r.u., relative units. The graph shows the height of peaks, corresponding to the postTC, relative to the peaks of the preTC.

Figure 4.

Effect of poly(A) tail length on peptide release. (A) Western blot analysis of preTCs-Nluc obtained at capped and uncapped mRNA containing poly(A) tails of different length with antibodies specific to PABP, rpL9, eRF1, eRF3a, eIF4A, eIF4B, eIF4E, eIF4G, eIF3a and eIF3b. As a control, 0.1 pmol of the corresponding purified protein was used. α means antibodies used to detect translation factors. (B) Rate of peptide release v₀ on uncapped preTC-Nluc in the absence and presence of exogenous PABP. RLU, relative luminescent units. The graphs show the mean ± standard error, n = 3. Asterisks indicate statistically significant differences between the values of different curves (*, P < 0.05; **, P < 0.01; ***, P < 0.001), while insignificant differences are not shown.

Figure 5.

Effect of poly(A) tail length on the kinetic constants of peptide release. (A) Schematic representation of the model mRNAs encoding nanoluciferase used in Nluc-preTC assembly. (B) Dependence of the kinetic constants apparent K_M, v_max and k_cat/K_M on the length of poly(A) tail. The graphs show the mean ± standard error, n = 3. Asterisks indicate statistically significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001), while insignificant differences are not shown. Red asterisks indicate differences between the values of different curves, blue and orange asterisks indicate differences with the A0 value of the corresponding curve.

Figure 6.

Model of the influence of the poly(A) tail length on the translation and closed-loop structure formation. (1) The first PABP molecule binds with the cap-bound eIF4F, promoting 43S PIC loading on the mRNA and 48S initiation complex formation. (2) The 48S initiation complex joins the 60S ribosomal subunit on the start codon, forming the 80S ribosome. eIF4F dissociates from the ribosome but remains bound to PABP on the poly(A) tail. (3) 80S ribosomes, while moving along the mRNA, unwind its secondary structure and maintain it in the untwisted state. Upon reaching the stop codon, the ribosome interacts with the eRF1-eRF3a complex, bound with PABP on the poly(A) tail. Release factors induce the hydrolysis of peptidyl-tRNA. (4) The 75 nt poly(A) tail is bound with three PABP molecules, forming a double closed-loop structure where the first PABP molecule interacts with the 48S initiation complex, and the third PABP molecule interacts with the terminating 80S ribosome. In such a structure, the ribosomal complexes are adjacent to each other, enhancing translation. Created with BioRender.com.